ALTERNATIVE STEM OF MODIFIED ANKARA VACCINIA VIRUS (MVA) The present invention relates to two new strains of modified Ankara Vaccinia virus (MVA) which has a greatly reduced virulence for most mammals, particularly humans, but which nonetheless grows in cells of a continuous line of cells approved for the production of therapeutic agents such as a vaccine. The invention also relates to a method for producing the aforementioned MVA-adapted strains. The MVA can be used, for example, for parenteral immunization, as a vector system, or in active or inactivated form as an adjuvant or as a regulator of non-specific components of the immune system. Background of the invention An organism is constantly being challenged by infectious agents such as bacteria, viruses, fungi or parasites. The immune system protects the body from permanent infections caused by these agents by destroying and eliminating these infectious agents and any toxic molecules produced by them. The immune system can be divided into a specific part and a non-specific part, although both parts are closely intertwined. The immunological - or specific response makes possible an immediate defense against a wide range of foreign substances and
Infectious agents. In contrast, the specific immune response is raised after a delay phase, when the organism is challenged with a substance for the first time. However, the specific immune response is highly efficient. The specific immune response is responsible for the fact that an individual recovering from a specific infection is protected against that specific infection but still vulnerable to other infectious diseases. In general, a second infection with the same infectious agent or a very similar one causes much lighter symptoms or no symptoms at all. Immunity persists for a long period, in some cases for life. This immunological memory is used for vaccination, where the organism is challenged by a harmless agent or an inactivated form of it, to induce a specific immunity. Sometimes adjuvants are incorporated into the vaccine to improve the specific immune response. A large part of the knowledge about infectious diseases and immunity is contributed by studies of smallpox. This disease is caused by variola virus, a member of the Orthopox virus species. Preventive inoculation with smallpox was initiated almost two centuries ago, resulting in immunization against smallpox. Subsequent immunization is
carried out with Vaccinia virus. In the early 1950s, many industrialized countries had eliminated endemic smallpox using vaccination with Vaccinia virus. However, vaccinating against smallpox with this Vaccinia virus occasionally caused serious complications, such as postvaccinal encephalitis, generalized vaccinia or contact infection. A new vaccine that does not show these complications was developed by Antón Mayr. The pox vaccine consists of the modified Ankara Vaccinia Virus (MVA) with poxvirus and was used for parenteral vaccination against smallpox in approximately 150,000 vaccinations without causing any complications related to vaccination. Not even children with immunological deficiencies showed serious side effects. The MVA was obtained by mutation and selection of the original Ankara vaccinia virus after 575 passages in chicken embryo fibroblast cultures. The safety of this MVA is reflected in biological, chemical and physical characteristics. MVA has a reduced molecular weight, six deletions in the genome, and is highly attenuated for mammalian cells, that is, DNA and proteins are synthesized, but virtually no viral particles are produced. The modified Ankara vaccinia virus developed by Antón Mayr was deposited in the Collection
European Cell Culture Center (ECACC), Salisbury, United Kingdom, under deposit number V94012707. Vaccination against smallpox was highly successful. In 1979, the World Health Organization declared the eradication of smallpox. Consequently, mass vaccination of children was discontinued and only laboratory workers and members of the armed forces of some countries were vaccinated. With the eradication of smallpox, the predominant cause of pox infections in humans was eliminated. However, some non-human poxviruses have a reduced host specificity, that is, they can cause infections not only in their typical host (for example cow for vaccinia), but also in other animals. As parts of the population are no longer immune to smallpox, infections with orthopox from animal species can be dangerous for them. Domestic animals are the main source of infections for humans. Consequently, the vaccination of domestic animals against orthopox virus is of increasing importance. Additionally, MVA may be of importance as a vector for gene therapy, ie, to transfer nucleic acid sequences to target cells where they are expressed. For a logarithmic reproduction of the MVA,
require cultures of primary or secondary chicken embryo fibroblast cells. The cells are obtained from chicken eggs incubated for 10 to 12 days. As the eggs are subject to biological variability, the cells obtained for the cell culture system are variable at a cellular level as well. In addition, in a "fibroblast culture" of chicken embryos, other cell types such as epithelial cells are often found. This variation of the cells results in a variation in the viruses produced in chicken embryo fibroblasts. Therefore it is difficult to standardize and validate a cell culture system to guarantee a consistently high quality of the MVA produced. In addition, contamination of a cell culture system with microorganisms or viruses already present in the incubated egg can not be completely excluded. When the MVA is cultured cells contaminated with virus, the MVA can recombine with the contaminating virus. In this way, an MVA can be generated with new and unpredictable characteristics. For large-scale virus production in a suspension culture, primary or secondary chicken embryo fibroblasts are also not highly suitable. Additionally, the purification and concentration of MVA by ultra-centrifugation of gradients would be advantageous. However, such purification is difficult, when MVA is grown
in primary or secondary fibroblasts of chicken embryos. Finally, an increasing number of patients have developed allergies against chicken egg albumen. Even when in vitro conditions of cultivation reduce the allergic potential strongly, the risk of an allergic reaction can be completely excluded. In sum, on the one hand the MVA can be cultured efficiently only in primary or secondary chicken embryo fibroblasts, causing a series of disadvantages, however, on the other hand, the safe application of the MVA in humans has been shown by its large-scale application as a vaccine. OBJECT OF THE INVENTION It is an object of the present invention to provide conditions for the homogeneous production of MVA virus particles. Additionally, the mentioned conditions should allow a simple and large-scale production of MVA. DETAILED DESCRIPTION OF THE INVENTION In order to achieve the foregoing and other objects, the invention provides a strain of MVA that is adapted for cell culture of a continuous cell line, wherein the cell line mentioned is approved for the production of an agent. therapeutic. In accordance with the present invention, at a time
First, efficient and large-scale production of MVA is possible. Since cells of a continuous line of cells are homogeneous and their characteristics stable, the MVA yield of these cell lines is also homogeneous with highly predictable characteristics. Additionally, the risk of contamination with microorganisms can be controlled and contamination of the MVA preparation with chicken egg proteins - as found when culturing MVA in chicken embryo fibroblasts - can be excluded. The handling of a permanent line of cells is convenient and in such a way highly advantageous for industrial application. In a preferred embodiment of the invention, the MVA is adapted for cell culture of a mammary cell line, which is approved for the production of a vaccine. It has been surprisingly found that MVA adapted to a mammary cell line such as the Vero cell still has a reduced virulence for humans and also for a wide range of other mammals. Consequently, the MVA is highly attenuated, that is, DNA and protein are synthesized but virtually no virus particle is produced, resulting in a virtually eliminated ability to cause the disease. Therefore, MVA according to the present invention is also highly advantageous as a vaccine for humans
and a wide range of mammals. As a result, the MVA is applicable particularly in the veterinary field. In addition, a method for obtaining a MVA strain according to the present invention is offered. In accordance with the embodiment of the invention, cells of a cell line that is approved for the production of a therapeutic substance are infected with a wild type of MVA. Preferably a multitude of infections (MOI) is used, i.e. a high amount of virus per cell is used for this infection. Subsequently, the viruses are collected and new cells from the same cell line are infected with the newly produced viruses. The mentioned procedure is repeated (serial passage), until the MVA has adapted to the aforementioned cell line. Adaptation is achieved, when at 72 hours after infection, the level of virus antibodies is at least 1 to 9 times, preferably 10 to 99 times, more preferably 100 to 106 times, and even more preferably more than 107 to 10. 1010 times increased compared to the level of entry virus antibodies. Adaptation is reached after a limited number of passages. "Adapted for growth" means that the amount of virus produced from an infection (output) is greater compared to the amount of virus originally used to infect the cells (input). In this
If the ratio of output / input is greater than 1. "Derivative" of the MVA deposited at ECACC, Salisbury, UK under depositary No. 99101431 and / or number of provisional access 01021411 denotes an MVA which is adapted for growing in Vero cells to a ratio that is essentially the same growth rate of the deposited strain, but which carries at least one difference in its genome compared to the deposited strain. The term "immune system" basically describes a complex involved in the defense of the organism against foreign substances and microorganisms. It is divided into a cell part comprising several cell types, such as for example, lymphocytes and other cells derived from white blood cells, and a humoral part comprising peptides and proteins such as antibodies, complement factors and cytokines. The term "immune response" describes the reaction of the immune system when a foreign substance or microorganism enters the body. Generally, the immune response is divided into a specific reaction and a nonspecific one, although both are closely intertwined. The non-specific immune response is considered as the immediate defense against a wide variety of foreign substances and infectious agents. The immune response
Specifically, it can be characterized as a highly efficient defense mechanism of the organism against a foreign substance that is generated against the aforementioned substance after a delay phase and that is highly specific for the aforementioned substance. The specific immune response is responsible for the phenomenon that an individual who has recovered from a specific infection is protected against this specific infection in the future. "Immune system activator" denotes any substance capable of causing or improving an immune response. "Immune system suppressant" denotes any substance capable of reducing or inhibiting an immune response. "Stabilizer of the immune system" denotes any substance capable of maintaining the immune response at a constant level. The inventors offer two preferred MVA strains that are adapted to an African green monkey cell line, called the Vero cell line (ATCC No. CCL-81). MVA strain, which has been passed 100 times in Vero cells was called "Vero-MVA" and deposited at the European Collection of Cell Cultures, Salisbury, UK, under deposit number 99101431. The MVA strain
after 200 passages in Vero cells was called "Vero-MVA-200" and deposited at ECACC under provisional accession number 01021411. The MVA obtained according to the above described is further expanded by culturing cells line approved cells under appropriate conditions, infecting cells with MVA and collecting the viral particles produced by said cells. Therefore, the MVA can be expanded efficiently and easily on a large scale. Surprisingly, the MVA of the invention does not show increased virulence in cells other than Vero cells, such as human cell lines, including HL, HEP-2 or HeLA. In another embodiment of the invention, the MVA contains at least one heterologous nucleic acid sequence, ie, a nucleic acid sequence that is not naturally found in the MVA genome (recombinant MVA). Preferably, the heterologous nucleic acid sequence is a gene, more preferably a gene encoding an immunization protein, and the most preferred one encodes an immunization protein against malaria, hydrophobia and / or hepatitis. The expression of the heterologous nucleic acid sequence mentioned is preferably under the transcriptional control of a vaccinia virus promoter, more preferably a
promoter of the MVA. In another preferred embodiment of the invention, the heterologous nucleic acid sequence is inserted into a deletion site that occurs naturally in the MVA genome (reported in PCT / EP96 / 02926). The recombinant MVA is used for the introduction of a nucleic acid sequence into a target cell, the nucleic acid sequence mentioned being homologous or heterologous to the target cell. The introduction of a heterologous nucleic acid sequence into a target cell can be used to produce heterologous nucleic acids, peptides and / or polypeptides and / or proteins encoded by the nucleic acid sequence in vitro. This method comprises the infection of host cells with recombinant MVA, the culture of the infected host cell under suitable conditions and optionally the isolation and / or enrichment of the peptide and / or the protein produced by the aforementioned host cell. Additionally, the introduction of a homologous or heterologous sequence can be applied to gene therapy in vitro and preferably in vivo. For in vitro and ex vivo gene therapy respectively, the cells are isolated from the individual by being treated, transformed with recombinant MVA and reintroduced to the individual from which the cells had been taken. For gene therapy in vivo, the
Recombinant MVA is administered directly to the living body of the animal including the human body. In a preferred embodiment of the invention, the recombinant MVA expresses an antigen or an antigenic epitope. More preferably, said vector expresses an antigenic determinant of Plasmodium falciparum, Mycobacteria, herpes virus, influenza virus, hepatitis, or human immunodeficiency virus. Since the MVA according to this invention is-surprisingly-still highly attenuated, the MVA is ideal for immunizing a wide range of mammals including humans. Accordingly, the present invention also provides a vaccine comprising the MVA for immunization of the living animal body including a human, against pox infections, preferably orthopox. The vaccine may additionally contain the MVA one or more additives such as an antibiotic, a preservative, or a stabilizer. The vaccine can be applied particularly in the veterinary field, for example, for the immunization of animals against orthopox infections such as cats against smallpox of cats, mice against ectromelia or camels against camelpox. The immunization is preferably done parenterally. The immunization effect of an antigenic determinant in a vaccine is improved many times by adding
a so-called auxiliary. An auxiliary co-stimulates the immune system in an unspecified manner provoking a stronger specific immunological reaction against the antigenic determinant of the vaccine. In accordance with another embodiment of the invention, the MVA is used as an auxiliary to co-stimulate the immune response against the antigenic determinant of the vaccine. In this case it is preferred that the MVA is deactivated. The deactivation of the MVA can be effected, for example, by heat or chemical substances. Preferably, the MVA is deactivated by β-propiolactone. In accordance with this embodiment of the invention, the deactivated MVA can be added to the vaccine against a number of infectious diseases to increase immunity against that disease. In the case of an infection, the immune, nervous, hormonal and vascular system of an individual collaborate closely. These interactions can be regulated by elements of the non-specific immune system, for example, cytokines such as interferons and interleukins. Pox-viruses can influence the regulation of the immune system (Swiss Vet 11/99, 13-17). As a result, in a further embodiment of the invention, the MVA and preferably the deactivated MVA is used in mammals including humans for
regulate the cellular and humoral elements of the non-specific (innate) immune system. Preferably, the MVA is used as a bioregulator, as immune system dysfunctions are eliminated and the body's own defenses are activated, stabilized and / or suppressed. More preferably, the MVA is used as a bioregulator in the case of a viral infection with, for example, herpes, hepatitis B or C virus, in the case of a chronic inflammatory disease and / or to support tumor therapy. The MVA can also be used to stabilize the immune system in a situation of increased susceptibility to infections such as stress or in newborns. The active or preferably deactivated MVA can be applied systematically, for example, intramuscularly and / or locally, for example, through mucous membranes and / or the skin. In conclusion, the present invention offers MVA strains that can be used in general for the same applications as the wild-type MVA, but eliminates the problems caused by the amplification of the wild-type MVA in chicken embryo fibroblasts. SUMMARY OF THE INVENTION The invention, among others, comprises the following, alone or in combination: A modified Ankara vaccinia virus (MVA)
adapted for the growth in cells of a continuous cell line, being that the aforementioned cell line is approved for the production of a therapeutic substance. The MVA according to the foregoing for cultivation in cells of a mammalian cell line. The MVA according to the preceding, being that the cell line is approved for the production of a vaccine. The MVA according to the preceding, being that the approved cell line is a Vero cell line. The MVA according to the preceding, being that the approved cell line is Vero cell line ATCC No. CCL-81. The MVA in accordance with the foregoing, deposited in the European Collection of Cell Cultures, Salisbury, United Kingdom under deposit No. 99101431 and / or its derivatives. The MVA in accordance with the foregoing, deposited at the ECACC, Salisbury, United Kingdom, under the provisional access number 01021411 and / or its derivatives. The MVA according to the preceding, comprising at least one heterologous nucleic acid sequence.
The MVA according to the foregoing comprising a heterologous nucleic acid sequence encoding, for example, for a therapeutic protein and / or an antigenic determinant such as a peptide immunizing against infection with malaria, hepatitis and / or hydrophobia. A host cell infected by the MVA described above. A composition, preferably a pharmaceutical composition, comprising the MVA described above and / or the DNA of the MVA. The pharmaceutical composition according to the above described, being that the pharmaceutical composition is a vaccine. The vaccine described above for the immunization of a living animal body including a human. The vaccine according to the foregoing for immunization against an infection with orthopox. The vaccine according to the foregoing for the immunization of cats against infection with smallpox, mice against infection with ectromelia and / or camels against infection with camel pox. The pharmaceutical composition according to the above described, being that the MVA is an activator, suppressor and / or stabilizer of the system
non-specific immunological A pharmaceutical composition comprising the MVA described above and / or the DNA of the MVA as an auxiliary. A pharmaceutical composition comprising the recombinant MVA described above and / or the recombinant MVA DNA. The pharmaceutical composition according to the above described for use in gene therapy. A method for introducing a homologous and / or heterologous nucleic acid sequence into a target cell comprising the infection of the target cell with the MVA described above. A method to obtain a MVA strain according to the previously described, comprising a) infection of cells of an approved cell line with a wild-type MVA, preferably the MVA deposited in ECACC under deposit No. V94012707, b) collecting the viruses, c) infection of new cells of the same line of cells with the newly produced viruses, and, optionally, d) repetition of b) and c) until the virus is adapted to the culture in the cells of the aforementioned cell line. A method for the production of viral particles of the MVA described above, comprising
cultivation of the cells of an approved cell line under appropriate conditions, infecting the aforementioned cell line with the aforementioned MVA, and collecting the viral particles produced by the aforementioned cells. The method according to the above described, being that the aforementioned cell line is infected with the MVA deposited in ECACC under the deposit No. 99101431 and / or the MVA deposited in ECACC under the provisional access number 01021411 or a derivative of one of these know. A method for producing a nucleic acid sequence, a polypeptide peptide and / or a protein, comprising infecting a host cell with the recombinant MVA described above, culturing the infected host cell under suitable conditions, and, optionally, isolating and / or enrichment of the nucleic acid, peptide and / or protein sequence produced by the aforementioned host cell. Use of the MVA described above for the production of a pharmaceutical composition for the treatment or prevention of a disease or a disorder sensitive to the aforementioned MVA. Use of the MVA described above for the production of a vaccine for the immunization of a living animal body including a human.
Use of the MVA described above for the production of an activator, suppressor and / or non-specific immune system stabilizer. The use according to the previously described for the manufacture of an auxiliary. Use of the MVA described above as a vaccine. use of the MVA described above as an auxiliary. Use of the MVA described above as an activator, suppressor and / or non-specific immune system stabilizer. A method for immunizing a living animal body including a human, wherein said method comprises administering a therapeutically effective amount of the above-described pharmaceutical composition to a person in need thereof. A method for introducing a nucleic acid sequence into a target cell comprising infecting the target cell with the MVA described above and / or the MVA DNA. A method for activating, suppressing and / or stabilizing the immune system of a living animal body including a human, wherein said method comprises administering the composition
pharmaceutical described above to a living animal body including a human. A method for improving the specific immune response against an antigenic determinant in a vaccine, comprising administering the MVA described above as an adjunct to a living animal body including a human. A modified Vaccinia Ankara virus adapted for cell culture of a continuous cell line by a method comprising the following steps: infect cells of a cell line being approved for the production of a therapeutic substance, collect the viral particles produced by the lines of mentioned cells and optionally, repeating the preceding steps until obtaining the desired growth characteristics of the mentioned MVA in the mentioned cells. EXAMPLES The following examples will illustrate the present invention more closely. It will be well understood to a person skilled in the art that the examples offered can in no way be interpreted in a manner that limits the applicability of the technology offered by the present invention to these examples. Example 1: Adaptation of MVA to Vero cells and
characterization of the aforementioned MVA strain 1. Adaptation of the MVA to Vero cells The wild-type MVA developed by Antón Mayr which is a modified Ankara Vaccinia virus was deposited in ECACC under N. deposition V 94012707. The wild-type MVA was adapted for growth in Vero cells by passage in series in virus in vero cells (table 1). The cell clone ATCC-No. CCL-81 from the stationary Vero cell line (WHO seed deposit ECACC No. 88020401) was used in passages No. 148 to 165 (WHO seed lot, master bank and work bank). The cells were propagated in a medium consisting of Eearle MEM (ICN), pH 7.4-7.6 and 5% of the BMS serum substitute (Biochrom). In accordance with a technique known to the people skilled in the art, the same cells of the workbench were always seeded dividing the cells 1: 2 to 1: 4. The medium contained approximately 250,000 cells per ml. The cells were respectively propagated in tubes (2ml), Roux dishes (100ml) and plastic dishes (6 and 40ml respectively). In general, the cells formed a simple co-luting layer after 16 to 24 h. Subsequently, the medium was replaced by simple Earle MEM without some additives. For the adaptation of the wild-type MVA a tube culture system was used. The result of the tickets
is summarized in Tables 1 and 2. Vero cells were infected with 10 MOI (multiplicity of infection) of the wild-type VA, that is on average, 10 viral particles per Vero cell. The wild-type MVA to start with was a genetically homogeneous MVA, purified from plaque after 575 passages in chicken embryo fibroblasts (antibody level: 107"75 KID5o / ml.) After 24h, 90% of the Vero cells the simple confluent layer were destroyed by toxic processes (50% by toxicity, 40% by decomposition) .The medium plus the debris of the cells after freezing and thawing of the cells, containing the produced viruses, was collected and 0.2ml of this The mixture was seeded in the single layer of Vero cells in the culture tube (2nd passage) This procedure was repeated 200. After the third passage, no toxic effect was observed, while a slightly cytopathic effect was seen (CPE). ) characterized by the roundness of the cells and decomposition in a period of 4 to 6 days after infection (p.inf.) The level of virus antibodies was 101'0 KIDso / ml. The MVA in Vero cells had started, however, very inefficiently. After the fifth passage, a typical CPE was observed to be completed after 4 or 5 days p.inf. The level of antibodies of the virus increased from 101'0 KIDso / ml
after the third passage at 104-0 KID50 / ml after the fifth passage. Therefore, the virus expanded more efficiently in Vero cells. In passages 5 to 11, a complete CPE was observed earlier and earlier and the antibody level of the virus increased in each passage. In passage 11 a stable level was reached at 107.5 KID50 / ml. Consequently, after eleven passages the adaptation of the MVA to the Vero cells had been achieved. In the next 30 additional passages, the result was the same for all passages and highly reproducible: The CPE already started 24h p.inf. and all the cells were affected after three days p.inf. At that time, 20% of the Vero cells were round and 80% were decomposed. After 3 days p.inf, the level of virus antibodies was always around 107"75 KID50 / ml After the 15th passage, the viruses were always collected after two or three days p.inf. used only 1 MOI instead of 10 to infect the cells (Table 2) .In the following additional passages the growth characteristics of the MVA changed only lightly.Remarkably, the virus's optimal antibody level increased more and reached 1010 KIDso / ml in passage 200. In conclusion, the virus grows exponentially in Vero cells exponentially.The growth characteristic mentioned is surprisingly different from
the characteristics of wild-type MVA. Consequently, a new strain of MVA was obtained through serial passages. The new strain mentioned was called "Vero-MVA" and after the passage 200 in vero cells "Vero-MVA-200". The Vero-MVA and Vero-MVA-200 were grown in larger quantities. For storage, the Vero-MVA was concentrated by centrifugation, resuspended in 2.5% polygeline and lyophilized in 2 ml flasks. The level of antibodies after lyophilation was still at least 108.5 KID50 / ml. The Vero-MVA and Vero-MVA-200 lyophilized were checked for contamination and toxicity and stored at + 4 ° C. 2. Characterization of the biological properties of Vero-MVA The biological characteristics of Vero-MVA
(passage 100) and Vero-MVA-200 (passage 200) were compared with the characteristics of wild-type MVA (table 3 and table 5). For this the techniques known by expert professionals were applied. The inventors showed that neither the host range of the virus was changed, except for the vero cells, nor was the virulence increased for humans or animals. Vero-MVA is still characterized by abortive propagation in non-admitted host cells. The main identity of viral particles
of Vero-MVA compared to the viral particles of the Elstree strain of vaccinia virus was shown by cross-reactivity of antibodies generated against the Elstree strain. The Elstree strain is a vaccinia strain recommended by the WHO for smallpox vaccination. Rabbit hyperimmune polyclonal sera generated against the Elstree strain was added to the Vero-MVA. 100 KID50 / ml of Vero MVA were completely neutralized in a serum dilution of 1: 512. A double dilution of serum was necessary to neutralize the same amount of the Elstree vaccinia strain (1: 256). Consequently, Vero-MVA can still be efficiently neutralized with vaccinia immune serum. The Ver-MVA, the Vero.MVA-200 and the wild-type MVA were compared with a number of additional assays as indicated in Tables 3, 4 and 5. The inventors showed that the virulence of Vero-MVA and Vero- MVA-200 for mammals including humans was not increased compared to the wild-type MVA. It was further shown that the Vero-MVA and Vero-MVA-200 are not contagious or toxic to mammals including humans. Surprisingly, the Vero-MVA cell specificity was more or less identical to the specificity of the wild-type MVA except for the vero cells: Vero-MVA is amplified almost as inefficiently in cells of human cell lines
(see table 4: HL, HEP-2 and HeLa cells) as the wild-type MVA. Consequently, although human cells and green monkey cells in Africa are phylogenetically closely related, Vero-MVA did not have the ability to be amplified in human cells. In other trials, no significant differences were seen either. In addition, the physical, chemical and biological characteristics of wild type MVA and Vero-MVA-200 were compared (table 500). While the wild type MVA grown in chicken embryo fibroblast cell cultures has 3 deletions in the inverted left terminal region, the Vero-MVA-200 has 4 deletions in the left terminal region compared to the pox-virus genome such as It was originally isolated in Ankara. Consequently, passing the wild type MVA by Vero cells resulted in an additional suppression. Vero-MVA was used to immunize pets against infections with orthopox. The serum of the animals was collected and a neutralization test was carried out. The inventors showed that the animals produced antibodies in high dosages. Antibody dosages were stable over a period of at least 111 days. It was also shown that the antibodies were able to neutralize in vitro viral particles of the MVA in a reduction assay of
plates. In conclusion, Vero-MVA can be used as a vaccine against infections with Orthopox in domestic animals and in humans. Table 1: Adaptation of MVA to Vero cells
* only 1 MOI instead of 10 MOI are planted after the eleventh passage.
Table 2: Canibio of virus antibody levels during adaptation of MVA to Vero cells. Passage No. Gathered after Antibody level [days p. inf ] for my [loqio / ml]
1 1 < 2.0 2 3 2.0 3 5 1.0 5 5 4.0 8 4 6.5 11 3 7.5 18 2 8.0 19 2 7.75 20 3 8.0 25 2 7.75 29 2 7.75 30 3 7.75 31 3 8.0 45 2 7.75 51 3 7.75 60 2 8.0 66 2 7.75 68 2 8.0 75 3 8.0 100 2 8.0 200 2 10.0
Table 3: Comparison of wild-type MVA and Vero-MVA characteristics MVA Vero-MVA type markers (100 'Vero-MVA-200 salvage and CPE passage in rounding and rounding cultures rounding and rounding off and off. single layer (1 MOI of cells after cells after 5 cells after 3 to 5-day seeding (90% days (100% CPE) 5 days (100% CPE) CPE) Antibody level 8.0 KID50 / ml 107 · 75 KIDso / ml 1010 · 0 KID / ml of optimal performance Abyssal reproduction Si sive in systems
cell phones not supported or
Reduced virulence Yes Yes: non-virulent for humans and absolute animals Contagiousness No No No Character of the plaque No nodes No nodes No primary nodes in proliferation sinproliferation without proliferation chorion membrane necrosis necrosis necrosis allantois hemglutination negative negative negative (( chicken erythrocytes) inactivation by β-kinetic kinetic of first cinéteico first order first order to order
propiolactone 0.05% 0.05% 0.04-0.05%
Protective effect Yes Yes in challenge test of mouse VSV baby toxicity for N No Non-human and animal stimulation of Interferon a, IL-2 Interferon a, IL-2 Interferon a and c, cytokines and 12, CSA and 12, CSA IL -2 and 12, CSA
Activation of Yes Yes Yes, increased phagocytosis, natural killer cells and T-lymphocytes
Table 4: Reproduction rate in KID50 / ml of Vero-MVA and wild-type MVA in different cell culture systems [logio / ml] Vero-MVA culture system MVA wild-type cell 31 ° Vero passage (575 ° passage in primary fobroblasts of chicken embryo)
1) Vero (cells of 8.0 4.5 kidney of the green monkey of Africa fibroblasts 4.5 8.5 chicken embryo primary '2) HL (lung 3.0 2.5 human) 1,2) HEP-2 (carcinoma 3.0 2.5 of human epidermis) 1,2 ) HeLA (carcinoma 2.75 2.75 human cervix) 1,2) BHK (5.75 cells 5.25 hamster kidney) 1,2) MDBK (3.5 3.5 kidney cells, bovine) 1,2) PK-15 (3.25 cells 3.5 kidney) pigs) 1) Continuous cell line derived from tissue and species indicated in parentheses 2) Cell line obtained from the institute collection for medical microbiology in Munich, Germany.
Table 5: Comparison of wild-type MVA (572 ° passage in chicken embryo fobroblast (CEF)) with Vero-MVA-200 (200 ° passage in Vero cells) MVA wild-type marker Vero-MVA-200 Markers 3 deletions in the 4 deletions in the genetic terminal region terminal region
(comparison with left left pox-virus strain (repeat as isolated in inverted terminal) Ankara genome size Additional reduced reduction from 208 to 178kb genome size to 172kb 15% loss of 20% molecular weight loss of weight Molecular genome original orignal genome Loss of additional receptor loss of receptors, eg interferon for IL-? β activating markers activated cell activation cells increased T-T (CD4, CD8, CD25) cytotoxic lymphocytes increased NK cell activity activation NK cells reproduction additional abortive reduction in cells in range of mammalian hosts in (except cell culture systems BHK) cellular cytokine interferon A, IL-2, interferon ayy, IL-12 IL-1,2, IL-12 level of CEF : 109-5KID5o / ml CEF: 109-5KID50 / ml Vero cell antibodies: 104 · 0 Vero cells: 109 · 5 KID50 virus / ml KIDso / ml system Reduced activity m immunological activity of the system immune system immunological specific nonspecific virulence for low no human and animal
REVINDICATIONS 1. A modified Ankara vaccinia virus (MVA), characterized in that it is adapted for the growth in cells of a continuous cell line, the cell line mentioned being approved for the production of a therapeutic substance. 2. The MVA according to claim 1, characterized in that it is adapted for growth in cells of a mammalian cell line. 3. The MVA according to claim 1 or
2, characterized in that the cell line is approved for the production of a vaccine. The MVA according to one of claims 1 to 3, characterized in that the approved cell line is a Vero cell line. 5. The MVA according to claim 4, characterized in that the approved cell line is the Vero cell line ATCC No. CCL-81. 6. The MVA according to claim 5, characterized in that it is deposited in the European Collection of Cell Cultures, Salisbury, United Kingdom under deposit No. 99101431 and / or its derivatives. 7. The MVA according to claim 5, characterized in that it is deposited in the ECACC, Salisbury, United Kingdom, under the provisional access number 01021411
and / or its derivatives. The MVA according to one of claims 1 to 7, characterized in that it comprises at least one heterologous nucleic acid sequence. 9. The MVA according to claim 8, characterized in that it comprises a heterologous nucleic acid sequence, for example, a gene coding for a therapeutic protein and / or an antigenic determinant.
10. A host cell infected by the MVA according to one of the preceding claims 1 to
9. 11. A composition, preferably a pharmaceutical composition, according to one of the preceding claims 1 to 9, characterized in that it comprises the MVA and / or the MVA DNA. 12. The pharmaceutical composition according to claim 11, characterized in that the pharmaceutical composition is a vaccine. The composition according to claim 12, characterized in that it is for the immunization of a living animal body including a human. The composition according to claim 12 or 13, characterized in that it is for immunization against an infection with orthopox.
15. The composition according to claims 12 to 14, characterized in that it is for the immunization of cats against infection with cat pox, mice against infection with ectromelia and / or camels against infection with camel pox. 16. The pharmaceutical composition according to claim 11, characterized in that the MVA is an activator, suppressor and / or stabilizer of the non-specific immune system. 17. A composition, preferably a pharmaceutical composition, according to one of the preceding claims 1 to 9, characterized in that it comprises the MVA and / or the DNA of the MVA as an auxiliary. 18. A composition, preferably a pharmaceutical composition, according to claim 8 or 9, characterized in that it comprises the recombinant MVA and / or the recombinant MVA DNA. 19. The pharmaceutical composition according to claim 18, characterized in that it is for use in gene therapy. 20. A method for introducing a homologous and / or heterologous nucleic acid sequence into a meta cell according to claim 8 or 9, characterized in that it comprises the infection of the target cell with a MVA. 21. A method to obtain a strain of MVA from
according to one of the preceding claims 1 to 7, characterized in that it comprises a) infection of cells of an approved cell line with a wild-type MVA, preferably the MVA deposited in ECACC under deposit No. V94012707, b) collecting the viruses, c) infection of new cells of the same cell line with the newly produced viruses, and, optionally, d) repetition of b) and c) until the virus is adapted to the culture in the cells of the cell line mentioned . 22. A method for the production of viral particles of the MVA according to one of the preceding claims 1 to 9, characterized in that it comprises a) culturing the cells of an approved cell line under suitable conditions, b) infecting the line of cells mentioned with the mentioned MVA, and c) collecting the viral particles produced by the mentioned cells. 23. The method according to claim 22, characterized in that said cell line is infected with the MVA according to claim 6 or 7.
24. A method for producing a nucleic acid sequence, a polypeptide peptide and / or a protein, characterized in that it comprises a) infection of a host cell with the recombinant MVA according to claim 8 or 9, b) cultivation of the cell of infected host under suitable conditions, and, optionally, c) isolation and / or enrichment of the nucleic acid sequence, peptide and / or protein produced by the aforementioned host cell. 25. Use of the MVA according to one of the preceding claims 1 to 9, characterized in that it is for the production of a pharmaceutical composition for the treatment or prevention of a disease or a disorder sensitive to the aforementioned MVA. 26. The use according to claim 25, characterized in that it is for the production of a vaccine for the immunization of a living animal body including a human. 27. The use according to claim 25, characterized in that it is for the production of an activator, suppressor and / or non-specific immune system stabilizer. 28. The use according to claim 25, characterized in that it is for the manufacture of an auxiliary.
29. Use of the MVA according to one of claims 1 to 9 as a vaccine. 30. Use of the MVA according to one of claims 1 to 9 as an auxiliary. 31. Use of the MVA according to one of claims 1 to 9 as an activator, suppressor and / or non-specific immune system stabilizer. 32. A method for introducing a nucleic acid sequence to a target cell according to claim 8 or 9, characterized in that it comprises infecting the target cell with the MVA and / or the MVA DNA. 33. A modified Vaccinia Ankara virus (MVA) adapted for cell culture of a continuous cell line by a method characterized in that it comprises the following steps: a) infecting cells of a cell line being approved for the production of a therapeutic substance, b) collecting the viral particles produced by the mentioned cell lines and optionally, c) repeating the preceding steps until obtaining the desired growth characteristics of the mentioned MVA in said cells.